Cataract-causing αAG98R-crystallin mutant dissociates into monomers having chaperone activity

[摘要] Purpose: The G98R mutation inαA-crystallin is associated with autosomal dominant cataract in humans.We have reported that mutant G98R protein has substrate-dependentchaperone activity. Further studies on this G98R mutant proteinrevealed that mutant protein shows reduced oligomeric stability andaccelerated subunit dissociation at a low protein concentration. Thepurpose of present study was to investigate the chaperone function ofdissociated subunits of αAG98R-crystallin. Methods: Substitution of glycine witharginine at position 98 in human αA-crystallin was accomplished bysite-directed mutagenesis. The recombinant protein was expressed in E.coli cells and purified by chromatographic techniques. PurifiedαAG98R-crystallin was diluted to a concentration of 0.1 mg/ml in 50 mMphosphate buffer containing 150 mM NaCl (pH 7.2) and incubated at37 °C for 24 h. The monomeric subunits were isolated from theoligomers through 50 kDa cutoff filters. The monomers wereanalyzed by SDS–PAGE, mass spectrometry, and circular dichroismspectroscopy and characterized by multi-angle light-scattering methods.Chaperone activity was tested against four client proteins: citratesynthesis, alcohol dehydrogenate, bovine βB2-crystallin andovotransferrin. Results: Gel filtration studies showedthat αAG98R-crystallin oligomers dissociate readily into monomers.Subunits of αAG98R-crystallin, isolated either by size exclusionchromatography or filtration showed chaperone activity againstheat-denatured alcohol dehydrogenase, citrate synthase, bovineβB2-crystallin, and chemically denatured ovatransferrin. SDS–PAGEanalysis of the mutant protein incubated at 37 °C for 12 daysshowed autolysis, which was confirmed by matrix-assisted laserdesorption ionization time-of-flight mass spectrometry (MALDI TOFMS/MS) analysis of αAG98R-crystallin fragments recovered afterSDS–PAGE. Conclusions: The present study showsthat the G98R mutation in αA-crystallin produces unstable oligomersthat dissociate into active chaperone subunits. The chaperone activityof the dissociated subunits against four client proteins suggests thatthe αA-crystallin subunits are the minimal units of chaperone activity.

[学科分类] 生物化学/生物物理